Conjugation in Tetrahymena: Ultrastructure of the meiotic prophase of the micronucleus

European Journal of

Europ.]. Protisto!' 28, 434-441 (1992) November 20, 1992

PROTISTOLOGY

Conjugation in Tetrahymena: Ultrastructure of the Meiotic Prophase of the Micronucleus Yoshiko Suganuma Biological Laboratory, Nara Saho-jogakuin College, Nara, Japan

Hiroshi Yamamoto Department Anatomy, Nara Medical University, Nara, Japan

SUMMARY The early conjugation process of Tetrahymena thermophila was studied with particular attention to the fine structure of the "crescent" micronucleus. The stationary macro- and micronuclei are tied together by the fused outer nuclear membrane. Inside the micronucleus, thick chromatin threads form a dense network. The micronucleus becomes detached from the concavity of the macronucleus and swells and loosened chromatin threads form fine threads. Most threads are gathered together in the center to form a large chromatin body, bur some threads remain partly dispersed along the periphery. The micronucleus is elongated by extended microtubules below the nuclear membrane. Chromatin threads that are anchored to structures considered to be precursors of kinetochores (PKI) arc present in the area that is proximal to the joint region of a conjugant as well as ro the inner nuclear membrane at the distal end. These extend together with the microtubules and become dispersed. The PKIs extend their own microtubles to the proximal end, pulling the chromatin threads back into the distal area. In the fully extended U-shaped micronucleus, PKI-microtubules have disappeared, and kinetochores (KI) are located in the vicinity of the inner nuclear membrane. The chromatin threads have become detached from the KI and have reassembled to form a dense network in the distal area.

Introduction Ciliates are unicellular eukaryotes that have two different nuclei; somatic macronuclei and germinal micronuclei. During conjugation, the macronucleus disintegrates and is replaced by one differentiated from the meiotic products of the micronucleus. In ciliates such as Paramecium [14], Tetrahymena [2,9], and Colpidium [4J, a peculiar structure called the "crescent" is seen by light microscopy in meiotic prophase during conjugation. Electron microscopical observations of nuclear changes during binary fission of Blepharisma [6], Urostyla [11] and Paramecium [5J have shown that nuclear division proceeds with no loss of the nuclear membrane. This process is called inner nuclear division. There have been few electron microscopic studies on nuclear changes during meiotic 0932 -4739/92/0028-0434$3.50/0

division. The crescent nucleus of Tetrahymena thermophila, which extends to almost twice the length of the body, reverts to a round shape just before meiotic division. This is of special interest in terms of morphological dynamism and functional association with meiosis. Here we report ultrastructural changes in the micronucleus during meiotic prophase in T. thermophila.

Material and Methods The complementary mating types used were II and IV of strain B of Tetrahymena thermophila, which were provided by Dr. 1. Sugai of Ibaraki University. The cells were cultured at 26°C in axenic growth medium containing proreose peptone (2%), yeast extract (1%) and glucose (0.6%). Late log-phase cells (lOb © 1992

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Gusrav Fischer Verlag, Stuttgart

Conjugation in Tetrahymena . 435 cells/ml) were harvested and washed three times by low-speed centrifugation in conditioning medium containing KCI (0.008%), NaCI (0.2%), and CaCI 2 (0.012%), and cultured with starvation in this medium for 24 h at 26°C. Approximately equal amounts of competent cells were mixed to induce conjugation. The cells were examined at definite times after mixing by light and electron microscopy. Samples for electron microscopy were collected by low-speed centrifugation, and fixed for 30 min in freshly prepared 0.2 M phosphate-buffered (pH 7.4) fixative consisting of two parts of 0.6% glutaraldehyde, one part of 2% osmium tetroxide, and one part of 1.2 % potassium bichromate. The cells were dehydrated by rapid passage through a graded ethanol series, and were embedded in Epoxy resin containing 11 g Quetol 812, 6 g DDSA and 5.8 g MNA [15]. Ultrathin sections, cut with an LKB ultratome, were stained with 1 % aqueous uranyl acetate and lead citrate and examined with a JEM 1200 EX electron microscope.

Results The ultrastructures of the micronuclei of unpaired cells were examined 30 min after mixing two complementary mating types (Fig. 1). This time probably corresponds to the late co-stimulation period [12]. The micronucleus (MI) located within the concavity of the macronucleus (MA) is connected to the MA by a membrane bridge (MB). The MB is formed by partial fusion of the outer membranes of the MI and MA (arrows). The karyoplasms of the two nuclei are, therefore, separated by only a pair of inner nuclear membranes. Many ribosomes (R) are attached to the outer nuclear membrane. Chromatin (CT) is closely packed in thick threads with relatively high electron density, forming a reticulum. At this stage, the nuclear membrane differs from that at interphase in that it has extrusions away from the CT. Immediately after formation of a pair, the MI expands slightly, but still remains located in the concavity of the MA (Fig. 2). The MI and MA are now separated by cytoplasm with no membrane bridge. The nuclear membrane (E) has formed a typical paired structure that has a nuclear pore complex (P). In addition, the thick chromatin threads (CT) that constitute the major portion of the nucleus have become less dense, and an area of low electron density has formed between the chromatin and

nuclear membrane. The chromatin threads appear to be attached to the nuclear membrane by a thin filamentous structure (arrow). As the micronucleus expands away from the concavity in the macronucleus, the chromatin is loosened and disintegrated into thin spiral fibrils of about 15-nm thickness (Fig. 3), most of which form a large dense chromatin body (CB) located in almost the center of the nucleus. Between the CB and nuclear membrane, an area of low electron density has expanded extensively and is associated with loosened threads or scattered granular chromatin (C). As in the interphase nucleus, nuclear pores (P) are present in some places in the nuclear membrane. Along the inner nuclear membrane, many microtubules (NT) with an outer diameter of 25 nm are visible in cross-sections (Fig. 3). The MI, which has begun to elongate along the long axis of the cell, is shown in Figs. 4-6. It has a slightly pointed end on the side near the joint region [13] (proximal end, or PE pole) while its other end (distal end, or DE pole) is gently rounded. Inside the nucleus, nuclear microtubuIes (NT) extend longitudinally along the inner nuclear membrane. Microtubules extend from the PE to the DE pole. In the ovoid or elongated ovoid nucleus, one side of the microtubule is connected to the inner membrane of the PE pole, but the other side does not reach the DE pole. Within the nucleus opposite the DE pole there are many structures of relatively low electron density that are considered to be precursors of kinetochores (PKI). The chromati~ threads (C) and microtubular bundles (KT) extend from these structures toward the large chromatin body (CB) (Figs. 4,5). The PKI are presumed to develop later into the kinetochore (KI). Because the inner nuclear microtubules (NT) run along the longitudinal axis within the inner nuclear membrane and extend to the PE and DE poles, the micronucleus takes on a cylindrical shape with an expanded trunk and two discoidal ends (Fig. 6). Inside the nucleus the chromatin body has loosened and disintegrated into thin filamentous chromatin, and the associated granular or thread portions have scattered. At the DE pole, the threads and filamentous chromatin are connected to the inner nuclear membrane (arrows).

Figs. 1-11. Transmission electron micrographs of the nuclei of the "crescent" in Tetrahymena thermophila. Fig. 1. Cross section of a micronucleus about 30 min after mixing starved complementary mating types. The region spanned by two ~ arrows is a membrane bridge, X 44 000. - Fig. 2. Cross section of a micronucleus about one hour after conjugation. It is located in the concavity of the macronucleus. The dense chromatin-thread network in the center is narrowly separated from the nuclear membrane, x 35 000. - Fig. 3. The micronucleus, now detached from the macronucleus, has at its center a chromatin body from which threads disperse. Microtubules just below the inner nuclear membrane run parallel to the membrane, in which nuclear pores are visible, X 33 000. Fig. 4. Longitudinal section of an elliptically elongated micronucleus. Two types of microtubules are distinguishable; one type running ~ parallel to the nuclear membrane, and the other extending from a structural located near the proximal end (near the joint region) that is thought to be a precursor of the kinetochore (PKI). The dense chromatin body is at the distal end. The chromonema originate here and spread over the nucleus, x 24 000. - Fig. 5. Proximal regions enlarged to show the PKls with extending microtubules and chromatin threads, X 28 000. - Fig. 6. Longitudinal section of the elongated micronucleus including the spread chromatin threads. At the distal end, the chromatin threads are connected to the inner nuclear membrane (arrows), x 10000.

436 . Y. Suganuma and H. Yamamoto Abbreviations, all figures: C = chromatin; CB = chromatin body; CT = chromatin threads; DE = distal end; E = nuclear envelope; JR = joint region; KI = kinetochore; KLS = kinetochore-like-structure; KT = kineto-microtubule; MA = macronucleus; MB = membrane bridge; MI = micronucleus; NT = nuclear-microtubule; P = nuclear pore; PE = proximal end; PKI = precursors of kinetochore (kinetochore-like-structures); R = ribosome.

Conjugation in Tetrahymena· 437

Conjugation in Tetrahymena . 439

Fig. 11. Proximal and distal ends of a micronucleus fully elongated into a U-shape in one of the pair members partitioned by the paired surface with cytoplasmic pores. The proximal end has Kls, and the distal end has concentrated chromatin threads attached at their ends (arrows) to the nuclear membrane. Microtubules can still be seen along the nuclear membrane, X 32 000.

The elongated cylindrical nucleus is occupied predominantly by thin filamentous chromatin (C) that, on the whole, is less electron dense (Figs. 7-10). Microtubules extend just below the inner nuclear membrane. During this stage, the micronucleus at PE is filled with kinetochores (KI) together with bundles of kinetochore-microtubules (KT) (Fig. 7), whereas the DE is filled with nuclear microtubules (NT). A large number of dispersed chromonemata (C) are attached to the inner nuclear membrane (Fig. 8). As the nucleus elongates further to a tubular shape, a number of Kls can be seen on the PE side (Figs. 9, 10). The kinetochores are about 210 nm in outer diameter, 150 nm long, and 20 nm thick and have a

cup-like form, the side of which points toward the PE. The alignment of the kinetochores is irregular. Many are located near thePE, and a few much further away. The rear of the kinetochores (toward the DE) contains thin twisted threads of chromatin (C) (Figs. 9, 10) which are considered to be recoiling. A cross section of the PE and DE ends of the fully elongated micronucleus with a U-shape is shown in Fig. 11. The two ends are located near the joint region (JR). A number of Kls are present on the PE side, whereas there are concentrated chromatin threads (eT) on the DE side. The edges of these threads are attached to the nuclear membrane of the DE at various points (arrows).

." Fig. 7. At the proximal end, microtubles extend from the KI, x 23 000. - Fig. 8. At the distal end, only nuclear membrane-associated microrubules are visible. Chromatin at the distal end is attached to the nuclear membrane (arrows), x 26 000. - Fig. 9. The elongated micronucleus, including Kls. The convex KI surfaces face the proximal end, at which the microtubules beneath the nuclear membrane are cut on the slant. Chromatin threads are thought to be pulled back to the distal end, x 31000. - Fig. 10. Kls localized at the proximal end. Microtubules run parallel to the nuclear membrane. Dispersed chromatin is visible, X 26 000.

440 . Y. Suganuma and H. Yamamoto

Discussion The meanings of crescent formation and the mechanism of its formation are discussed below on the basis of ultrastructural observations on meiotic prophase of Tetrahymena thermophila. 1. Nuclear Membrane

Thirty minutes after mixing complementary mating type cells, the nuclear membrane of the micronucleus in the unpaired cells puts out an extrusion, the basic structure of which is similar to that of the nucleus at interphase, which forms a typical unit membrane containing a nuclear pore complex. The micro- and macronuclei are connected by a membrane bridge formed by the fusion of their outer membranes. There is no structure similar to the cytoplasmic microtubules that connect the nuclear pores of the micro- and macronuclei of P. multimicronucleatum [5]. The extrusions seen on some parts of the nuclear membrane suggest that the membrane surface has already begun to increase at this stage when the micro- and macronuclei are connected by a membrane bridge. After the conjugant forms, the micronucleus shows a tremendous increase in membrane surface during its expansion and elongation. Structures such as the endoplasmic reticulum or vesicles are considered to contribute to increasing the membrane surface, but no incorporation of these structures into the nuclear membrane was observed at any stage of crescent formation. Ribosomes densely distributed in the cytoplasm of the outer membrane would ensure supply of membrane materials. Because the nuclear membrane maintains an unchanged basic structure, meiotic division is taken to be inner nuclear division as is mitotic division at binary fission. 2. Microtubules

During crescent formation, the inner nuclear microtubuies can be classified as two types on the basis of their origins and functions: a) Microtubules below the nuclear membrane. Micronuclear expansion starts at the proximal end, which is the joint region of a conjugant. The micronucleus changes from an egg shape to an elongated egg shape as microtubuies parallel to and below the nuclear membrane elongate. When these microtubules reach the distal end, the micronucleus becomes cylindrical. On further elongation of the microtubules, however, it becomes U-shaped. The microtubules disappear in this U-shaped crescent micronucleus. That is, the microtubules below the nuclear membrane are considered to contribute to crescent formation by forming an inner nuclear skeleton. b) Microtubules of the kinetochore. In the early stages of nuclear elongation, bundles of microtubules as well as thread-like chromatin stretch from a structure (thought to be a precursor of the kinetochore) near the proximal end to

the large chromatin body. They disappear when the chromatin body disperses, leaving the nucleus filled with thin threads and filamentous chromatin. This occurs before the microtubules below the nuclear membrane disappear. The function of these microtubule-bundles is apparently to fix one end of the chromatin on the kinetochore, thus disassociating the chromatin as it elongates. 3. Chromatin and Kinetochore

Ray [9] reported that optical microscopic studies on cells stained by the aceto-carmine squash method, showing that the crescent of T. thermophila consists of chromosomes connected in vertical array. By the Giemsa method [1], Martindale [7] found that the crescent consists of paired (n = 5) 2-value chromosomes disposed in line. Wolfe [15] suggested that the elongated state of the micronucleus provides an opportunity for chromosomal crossing over. Sugai et al. [10], who used a spreading technique [8], proposed that most elongated crescents consist of tandemly connected chromatin threads paired longitudinally, each thread probably containing a single genome. Electron microscopic observations by Wolfe [16] showed that parallel strands of chromatin stretch out from the chromatin body along the long axis. Our observations clearly show that in the micronucleus during elongation, kinetochores associated with chromatin threads and microtubule bundles are formed near the proximal end and that the chromatin threads that stretch from the large chromatin body are inscribed on the nuclear membrane at the distal end. The large chromatin body is, therefore, probably stretched by elongation of the kinetochore microtubules and the microtubules below the nuclear membrane; i.e., by elongation of the nucleus. The presence of many kinetochores suggests that the thin filamentous chromatin disassociates into many fragments during this elongation. When nuclear elongation begins, many kinetochore-like structures of lower electron density than that of chromatin are present near the proximal end and are associated with microtubule bundles and loose threads of chromatin. But when the nucleus is filled with dispersed, thin filamentous chromatin, kinetochores that have a typical cap-like structure appear at various sites inside the elongated nucleus, eventually re-aggregating near the proximal end. Probably, kinetochores are always attracted to the proximal end. After their detachment from kinetochores, chromatin threads are attracted to the distal end, forming a concentration of chromatin threads that eventually regains a coarse reticular structure just before meiotic division. Throughout this reorganization of the chromatin, there is no evidence of the so-called long-axis disposition of chromatin. Moreover, there is no typical chromosome structure such as that seen in the meiotic metaphase of T. thermophila (unpublished) and in the binary fission of Urostyla [11]. Nor is there a structure similar to the synaptonemal complex seen in Blepharisma in meiotic prophase [3].

Conjugation in Tetrahymena· 441

Acknowledgements We thank Dr. Y. Takagi of Nara Women's University for his careful reading of our maniscript and helpful suggestions. We are also grateful to Miss N. Tamefusa for technical assistance and to Dr. T. Sugai, Ibaraki University, for providing the two types of T. thermophila used.

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7 Martindale D. W., Allis C. D. and Bruns P. J. (1982): Conjugation in Tetrahymena thermophila, a temporal analysis of cytological stages. Exp. Cell Res., 140, 227-236. 8 Prescott D. M. and Bender M. A. (1964): Preparation of mammalian metaphase chromosomes for autoradiography. In: Prescott D. M. (ed.): Methods in cell physiology, 1, pp. 381-384. Academic Press, N.Y. 9 Ray C. H. Jr. (1956): Meiosis and nuclear behavior in Tetrahymena pyriformis. J. Protozool., 3, 88-96. 10 Sugai T. and Hiwatashi K. (1974): Cytologic and autoradiographic studies of the micronucleus at meiotic prophase in Tetrahymena pyriformis. J. Protozool., 21(4), 542-548. 11 Suganuma Y. (1969): Electron microscope studies on the mitosis of binary fission of the micronucleus of the ciliate, Urostyla (in Japanese). J. Nara Med. Univ., 20, 323-335. 12 Suganuma Y., Shimode C. and Yamamoto H. (1984): Conjugation in Tetrahymena: formation of a special junction area for conjugation during the co-stimulation period. J. Electron Microsc., 33, 10-18. 13 Suganuma Y. and Yamamoto H. (1988): Conjugation in Tetrahymena: its relation to Concanavalin A receptor distribution on the cell surface. Zool. Sci., 5, 323-330. 14 Wichterman R. (1953): The biology of Paramecium. Blakiston, N.Y. 15 Wolfe J. (1973): Conjugation in Tetrahymena: the relationship between the division cycle and cell pairing. Dev. BioI., 35, 221-231. 16 Wolfe J., Hunter B. and Steven W. A. (1976): A cytological study of micronuclear elongation during conjugation in Tetrahymena. Chromosoma (Berl.), 55, 289-307.

Key words: Conjugation - Tetrahymena - Ultrastructure - Meiosis - Crescent Yoshiko Sugaruma and Hiroshi Yamamoto, Biological Laborator~,. Nara Saho-jogakuin College, Rokuyaon-Cho Nara, Japarr